Curcumin analogues and uses thereof

ABSTRACT

The present invention relates to compounds capable of acting as androgen receptor antagonists, pharmaceutical formulations containing the same, and methods of use thereof. Such uses include, but are not limited to, use as antitumor agents, particularly for the treatment of cancers such as colon, skin and prostate cancer and to induce androgen receptor antagonist activity in a subject afflicted with an androgen-related affliction. Examples of androgen-related afflictions include, but are not limited to, baldness, hirsutism, behavioral disorders, acne, and uninhibited spermatogenesis wherein inhibition of spermatogenesis is so desired.

RELATED APPLICATIONS

This application is a continuation-in-part of PCT InternationalApplication No. PCT/US2003/009350, filed Mar. 27, 2003, titled NovelCurcumin Analogues and Uses Thereof, published in English on Oct. 30,2003, which claims priority from U.S. patent application Ser. No.10/124,642, filed Apr. 17, 2002, titled Novel Curcumin Analogues andUses Thereof, now issued as U.S. Pat. No. 6,790,979, the disclosures ofwhich are incorporated by reference herein in their entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with Government support under Grant No. CA-17625and Grant No. CA-55639. The government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to compounds capable of acting as androgenreceptor antagonists, pharmaceutical formulations containing the same,and methods of use thereof.

BACKGROUND OF THE INVENTION

The androgen receptor (AR) is a member of a large family ofligand-dependent transcriptional factors known as the steroid receptorsuperfamily. Chang et al., Proc. Natl. Acad. Sci. USA, 85, 7211-7215(1988). Beato, M., Cell, 56, 335-344 (1989). Androgens and the AR playan important role in the growth of the normal prostate and prostatecancer. Prostate cancer represents the most common male malignancy inthe United States. Landis et al., Cancer J. Clin., 48, 6-29 (1998).Recently, antiandrogens such as hydroxyflutamide (HF) in combinationwith surgical or medical castration have been widely used for thetreatment of prostate cancer. Crawford et al., New Engl. J. Med., 321,419-424 (1989). Several compounds, including cyprosterone, HF, andbicalutamide (shown below), have been used clinically in the treatmentof prostate cancer.

The synthetic steroidal antiandrogen cyprosterone is one of the firstantiandrogens used clinically in Europe, McLeod, D., G., Cancer, 71,1046-1049 (1993) but it has many side effects. Neumann et al., J. Clin.Oncol., 1, 41-65 (1982). HF and bicalutamide are both nonsteroidalantiandrogens. Bicalutamide is a newer nonsteroidal antiandrogenoriginally thought to have a pure antiandrogen activity without agonistactivity. It has a longer half-life (6 days) and a higher bindingaffinity to the AR than HF. Verhelst et al., Clin. Endocrinol., 41,525-530 (1994). (a) Kelly et al., J. Urol. (1993), 149, 607-609; (b)Scher et al., Prostate Cancer. J. Clin. Oncol., 11, 1566-1572 (1993).

Although antiandrogen hormone therapy has been widely used for thetreatment for prostate cancer, some antiandrogens may act as AR agonistswhich may result in “antiandrogen withdrawal syndrome.” Miyamoto et al.,Proc. Natl. Acad. Sci. USA, 95, 7379-7384 (1998). A currently acceptedhypothesis postulates that mutations in androgen receptors may accountfor why HF, the active metabolite of flutamide, can activate androgenreceptor target genes and stimulate prostate cancer growth. Miyamoto etal., Proc. Natl. Acad. Sci. USA, 95, 7379-7384 (1998). The samemechanism is used to explain the “flutamide withdrawal syndrome,” inwhich patients who experience an increase in prostate-specific antigen(PSA) while taking flutamide, have a decrease in PSA after withdrawal oftreatment. Indeed, HF can activate androgen receptor target genes, suchas PSA and MMTV-LTR (a reporter gene which expressed androgen-responseelement), in the presence of ARA70, the first identified androgenreceptor co-activator. Yeh et al., The Lancet, 349, 852-853 (1997).Because this syndrome often leads to the failure of androgen-ablativetherapy, it is desirable to develop better antiandrogens without agonistactivity.

The phenolic diarylheptanoid curcumin (1) is the major pigment inturmeric. Curcumin and its analogs show potent anti-oxidant activity,anti-inflammatory activity, Nurfina et al., Eur. J. Med. Chem., 32,321-328 (1997) cytotoxicity against tumor cells, Syu et al., J. Nat.Prod., 61, 1531-1534 (1998), antitumor-promoting activities, Sugiyama etal., Biochem. Pharmacol., 52, 519-525 (1996). Ruby et al., Cancer Lett.,94, 79-83 (1995) and antiangiogenesis activity (J. L. Arbiser et al.Mol. Med. 4: 376 (1998)).

Two cyclic diarylheptanoids, 13-oxomyricanol and myricanone, exhibitingpotent antitumor promoting effects on DMBA-initiated and TPA-inducedmouse skin carcinogenesis have been reported. Ishida et al., CancerLett., 159. 135-140 (2000). In the present study, a number of novelcurcumin analogues have been prepared and evaluated for antagonisticactivity against the AR in the presence of androgen receptorcoactivator, ARA70, using two human prostate cancer cell lines, PC-3 andDU-145. PC-3 cells are androgen-independent tumor cells that do notexpress functional AR. DU-145 cells are androgen-independent tumor cellsthat also do not express functional AR.

SUMMARY OF THE INVENTION

According to embodiments of the present invention, the present inventionrelates to a compound according to formula I:

A first aspect of the present invention is a compound according toformula I or Ia:

wherein:

R′ and R″ are each independently selected from the group consisting ofH, alkoxy, and halo;

R_(a) and R_(b) are each independently selected from the groupconsisting of H, alkyl and halo;

R₁ and R₂ are each independently selected from the group consisting ofH, hydroxy, alkoxy, nitro, amino, and dialkylamino;

R₃ and R₄ are each independently selected from the group consisting ofH, hydroxy, halo, alkoxy, and —OR₇C(O)R₈, wherein R₇ is lower alkyleneand R₈ is alkoxy;

or R₁ and R₃ together are alkylenedioxy;

or R₂ and R₄ together are alkylenedioxy;

R₅ and R₆ are each independently selected from the group consisting ofH, halogen, nitro and alkoxy;

X₁ is N, or X₁ is C bonded to a H, alkoxy or nitro; and

X₂ is N, or X₂ is C bonded to a H, alkoxy or nitro;

subject to the proviso that curcumin is excluded therefrom;

or a pharmaceutically acceptable salt thereof.

A further aspect of the present invention is a compound according toformula II:

wherein:

R₁₁ and R₁₂ are each independently selected from the group consisting ofalkoxy, nitro, amino, and dialkylamino;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydroxy, alkoxy, —OR₁₈C(O)R₁₉ wherein R₁₈ is lower alkylene oralkenylene and R₁₉ is alkoxy, and tetrahydropyranyl;

or R₁₁ and R₁₃ together are alkylenedioxy;

or R₁₂ and R₁₄ together are alkylenedioxy;

R₁₅ and R₁₆ are each independently selected from the group consisting ofH, halogen, and nitro;

R₁₇ is —R₂₀C(O)OR₂₁, wherein R₂₀ is alkylene or alkenylene and R₂₁ is Hor alkyl;

X₃ is N, or X₃ is C bonded to a H, alkoxy or nitro; and

X₄ is N, or X₄ is C bonded to a H, alkoxy or nitro;

or a pharmaceutically acceptable salt thereof.

According to yet other embodiments of the present invention, the presentinvention relates to compounds according to the formula III:

-   -   wherein:        -   R₂₅ and R₂₆ are each independently H or lower alkyl;        -   R₂₇, R₂₈, R₂₉ and R₃₀ are each alkoxy;        -   R₃₁ is H or lower alkyl;    -   or a pharmaceutically acceptable salt thereof.

A further aspect of the present invention is a compound of Formula IV:

wherein:

R₄₁ and R₄₂ are each independently hydroxy or alkoxy; and

R₄₃ and R₄₄ are each independently hydroxy or alkoxy;

or a pharmaceutically acceptable salt thereof.

A further aspect of the invention is a compound of the Formula:

or a pharmaceutically acceptable salt thereof.

According to still other embodiments of the present invention, thepresent invention relates to a method of treating cancer, comprisingadministering to a subject in need thereof a treatment effective amountof a compound according to the formulas above or curcumin. Examples ofcancers that may be treated include, but are not limited to, skincancer, small cell lung cancer, testicular cancer, lymphoma, leukemia,esophageal cancer, stomach cancer, colon cancer, breast cancer,endometrial cancer, ovarian cancer, central nervous system cancer, livercancer and prostate cancer.

According to yet other embodiments of the present invention, the presentinvention relates to a method of inducing androgen receptor antagonistactivity, the method comprising contacting a cancer cell with anandrogen receptor antagonist effective amount of a compound according tothe formulas above or curcumin.

According to other embodiments of the present invention, the presentinvention relates to a method of inducing androgen receptor antagonistactivity in a subject afflicted with an androgen-related affliction byadministering a compound according to the formulas above or curcumin.Examples of androgen-related afflictions include, but are not limitedto, baldness, hirsutism, behavioral disorders, acne, and uninhibitedspermatogenesis wherein inhibition of spermatogenesis is so desired.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates structures of curcumin analogues (1-20);

FIG. 2 illustrates structures if curcumin analogues (21-44);

FIG. 3A illustrates suppression of DHT-mediated MMTV transcription ARactivity by hydroxyflutamide (HF) and selected compounds;

FIG. 3B illustrates suppression of DHT-mediated MMTV transcription ARactivity by hydroxyflutamide (HF) and selected compounds; and

FIG. 3C illustrates suppression of DHT-mediated MMTV transcription ARactivity by hydroxyflutamide (HF) and selected compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, which further illustrate theinvention described herein. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety.

The term “alkyl” or “loweralkyl” as used herein refers to C1 to C4, C6or C8 alkyl, which may be linear or branched and saturated orunsaturated.

“Curcumin” as used herein refers to a compound of the Formula:

Curcumin, or a pharmaceutically acceptable salt thereof, may be used tocarry out the methods described herein.

“Cycloalkyl” is specified as such herein, and is typically C3, C4 or C5to C6 or C8 cycloalkyl.

“Alkenyl” or “loweralkenyl” as used herein likewise refers to C1 to C4alkenyl, and alkoxy or loweralkoxy as used herein likewise refers to C1to C4 alkoxy.

“Alkoxy” as used herein refers to linear or branched, saturated orunsaturated oxo-hydrocarbon chains, including for example methoxy,ethoxy, propoxy, isopropoxy, butoxy, and t-butoxy.

The term “aryl” as used herein refers to C3 to C10 cyclic aromaticgroups such as phenyl, naphthyl, and the like, and includes substitutedaryl groups such as tolyl.

“Halo” as used herein refers to any halogen group, such as chloro,fluoro, bromo, or iodo. In some embodiments halo is preferably fluoro.

The term “hydroxyalkyl” as used herein refers to C1 to C4 linear orbranched hydroxy-substituted alkyl, i.e., —CH₂OH, —(CH₂)₂OH, etc.

The term “aminoalkyl” as used herein refers to C1 to C4 linear orbranched amino-substituted alkyl, wherein the term “amino” refers to thegroup NR′R″, wherein R′ and R″ are independently selected from H orlower alkyl as defined above, i.e., —NH₂, —NHCH₃, —N(CH₃)₂, etc.

The term “tetrahydropyranyl” refers to a group of the formula:

The term “oxyalkyl” as used herein refers to C1 to C4 oxygen-substitutedalkyl, i.e., —OCH₃, and the term “oxyaryl” as used herein refers to C3to C10 oxygen-substituted cyclic aromatic groups.

The term “alkylenedioxy” refers to a group of the general formula—ORNO—, —ORNORN—, or —RNORNORN— where each RN is independently alkyl.

“Treat” or “treating” as used herein refers to any type of treatmentthat imparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the disease, prevention or delayof the onset of the disease, etc.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

“Inhibit” as used herein means that a potential effect is partially orcompletely eliminated.

“Androgen” as used herein refers to sex hormones generally known tothose skilled in the art and include, but are not limited to,testosterone, dihydrotestosterone, and androstenedione and compoundsknown to act in mechanisms similar to androgens such as androgenreceptor agonists. “Androgen” relates to a hormone or compound, or acombination thereof.

“Antiandrogen withdrawal syndrome” as used herein refers to a phenomenoncharacterized by either no change or an increase in serumprostate-specific antigen (PSA) concentration upon administration ofantiandrogen therapy, and a subsequent decreased PSA concentrationobserved after withdrawal of antiandrogen therapy.

“Androgen receptor antagonist” as used herein refers to a compound thatpartially or completely inhibits the activity of an androgen receptoragonist.

“Androgen-related affliction” as used herein refers to conditionswherein an androgen or combination of androgens play a role in thecondition observed.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other animalsubjects (i.e., mammals, avians) for veterinary purposes. Mammals arepreferred, with humans being particularly preferred.

As noted above, a first aspect of the present invention is a compoundaccording to Formulas I or Ia:

wherein:

R′ and R″ are each independently selected from the group consisting ofH, alkoxy, and halo;

R_(a) and R_(b) are each independently selected from the groupconsisting of H, alkyl and halo;

R₁ and R₂ are each independently selected from the group consisting ofH, hydroxy, alkoxy, nitro, amino, and dialkylamino;

R₃ and R₄ are each independently selected from the group consisting ofH, hydroxy, halo, alkoxy, and —OR₇C(O)R₈, wherein R₇ is lower alkyleneand R₈ is alkoxy;

or R₁ and R₃ together are alkylenedioxy;

or R₂ and R₄ together are alkylenedioxy;

R₅ and R₆ are each independently selected from the group consisting ofH, halogen, nitro and alkoxy;

X₁ is N, or X₁ is C bonded to a H, alkoxy or nitro; and

X₂ is N, or X₂ is C bonded to a H, alkoxy or nitro;

subject to the proviso that curcumin is excluded therefrom;

or a pharmaceutically acceptable salt thereof.

In some particular embodiments of compounds of Formulas I and Ia, atleast one of R′ and R″ are alkoxy or halo.

In some particular embodiments of compounds of Formulas I and Ia, R_(a)is halo and R_(b) is H, alkyl or halo, preferably alkyl.

In some particular embodiments of comounds of Formulas I and Ia, R₁ andR₂ are each independently selected from the group consisting of alkoxy,nitro, amino, and dimethylamino.

In some particular embodiments of compounds of Formulas I and Ia, R₁ andR₃ together are methylenedioxy or ethylenedioxy.

In some particular embodiments of compounds of Formulas I and Ia, R₂ andR₄ together are methylenedioxy or ethylenedioxy.

In some particular embodiments of compounds of Formulas I and Ia, R₅ andR₆ are each independently selected from the group consisting of H, F,and nitro.

A further aspect of the present invention is a compound according toFormula II:

wherein:

R₁₁ and R₁₂ are each independently selected from the group consisting ofalkoxy, nitro, amino, and dialkylamino;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydroxy, alkoxy, —OR₁₈C(O)R₁₉ wherein R₁₈ is lower alkylene oralkenylene and R₁₉ is alkoxy, and tetrahydropyranyl;

or R₁₁ and R₁₃ together are alkylenedioxy;

or R₁₂ and R₁₄ together are alkylenedioxy;

R₁₅ and R₁₆ are each independently selected from the group consisting ofH, halogen, and nitro;

R₁₇ is —R₂₀C(O)OR₂₁, wherein R₂₀ is alkylene or alkenylene and R₂₁ is Hor alkyl;

X₃ is N, or X₃ is C bonded to a H, alkoxy or nitro; and

X₄ is N, or X₄ is C bonded to a H, alkoxy or nitro;

or a pharmaceutically acceptable salt thereof.

In some particular embodiments of compounds of Formula II, R₂₀ isalkenylene.

In some particular embodiments of compounds of Formula II, R₁₃ and R₁₄are tetrahydropyranyl.

In some particular embodiments of compounds of Formula II, R₁₁ and R₁₂are each independently selected from the group consisting of alkoxy,nitro, amino, and dimethylamino.

In some particular embodiments of compounds of Formula II, R₁₁ and R₁₃together are methylenedioxy or ethylenedioxy.

In some particular embodiments of compounds of Formula II, R₁₂ and R₁₄together are methylenedioxy or ethylenedioxy.

In some particular embodiments of compounds of Formula II, R₁₅ and R₁₆are each independently selected from the group consisting of H, F, andnitro.

According to yet other embodiments of the present invention, the presentinvention relates to compounds according to the formula III:

wherein:

R₂₅ and R₂₆ are each independently H or lower alkyl;

R₂₇, R₂₈, R₂₉ and R₃₀ are each alkoxy;

R₃₁ is H or lower alkyl;

or a pharmaceutically acceptable salt thereof.

A further aspect of the present invention is a compound of Formula IV:

wherein:

R₄₁ and P₄₂ are each independently hydroxy or alkoxy; and

R₄₃ and R₄₄ are each independently hydroxy or alkoxy;

or a pharmaceutically acceptable salt thereof.

A further aspect of the invention is a compound of the Formula:

or a pharmaceutically acceptable salt thereof.A. Specific compounds

Specific compounds within the scope of the present invention include,but are not limited to:

Additional examples of compounds of the invention are set forth below.B. Synthesis of Compounds

Variations on the following general synthetic methods will be readilyapparent to those skilled in the art and are deemed to be within thescope of the present invention.

FIGS. 1 and 2 show the structures of curcumin analogues and1,3-diaryl-1,3-diketopropane derivatives. Curcumin (1),demethoxycurcumin (2) and bisdemethoxycurcumin (3) were obtained bycolumn chromatography (silica gel, CHCl₃-MeOH) of commercially availablecurcumin (Aldrich), which contained 2 and 3 as minor components.Treatment of 1 with diazomethane gave dimethylated curcumin (4) andmonomethylated curcumin (9). Methylation of 1 with methyl iodide andK₂CO₃ furnished the trimethylated derivative 10, in which a methyl groupwas also introduced at the C-4 position. Compounds 58 were synthesizedby heating 1-4 with histidine hydrazide, AcOH and p-TsOH overnight.Hydrogenation of 1 with 10% Pd—C gave a mixture of 11-13. Similarly,compounds 14-16 and 17-18 were obtained by hydrogenation of 4, and 10,respectively. Heating 1 with methyl chloroacetate, NaI and K₂CO₃ inacetone furnished a mixture of monomethoxycarbonylmethyl ether 18 andbis-methoxycarbonylmethyl ether 19, which were separated by preparativeTLC (PLC). Compounds 21-23 were prepared from benzene or vanillin andethyl 4-acetyl-5-oxohexanoate by a method known in the art. Pedersen etal., Liebigs Ann. Chem., 1557-1569 (1985). Compounds 21-23 constitute anunseparable mixture of keto-enol tautomeric isomers. The syntheses of24-38 were described in our previous paper. Ishida et al., Cancer Lett.,159. 135-140 (2000). Ishida et al., Synthesis and Evaluation of CurcuminAnalogues as Cytotoxic Agents. Unpublished data. Compounds 39-44 werepurchased from Aldrich, Inc (Milwaukee, Wis.).

C. Pharmaceutically Acceptable Salts

The term “active agent” as used herein, includes the pharmaceuticallyacceptable salts of the compound. Pharmaceutically acceptable salts aresalts that retain the desired biological activity of the parent compoundand do not impart undesired toxicological effects. Examples of suchsalts are (a) acid addition salts formed with inorganic acids, forexample hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like; and salts formed with organic acids suchas, for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid,p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonicacid, and the like; and (b) salts formed from elemental anions such aschlorine, bromine, and iodine.

Active agents used to prepare compositions for the present invention mayalternatively be in the form of a pharmaceutically acceptable free baseof active agent. Because the free base of the compound is less solublethan the salt, free base compositions are employed to provide moresustained release of active agent to the target area. Active agentpresent in the target area which has not gone into solution is notavailable to induce a physiological response, but serves as a depot ofbioavailable drug which gradually goes into solution.

D. Pharmaceutical Formulations

Curcumin and the curcumin analogues of the present invention are usefulas pharmaceutically active agents and may be utilized in bulk form. Morepreferably, however, these compounds are formulated into pharmaceuticalformulations for administration. Any of a number of suitablepharmaceutical formulations may be utilized as a vehicle for theadministration of the compounds of the present invention.

The compounds of the present invention may be formulated foradministration for the treatment of a variety of conditions. In themanufacture of a pharmaceutical formulation according to the invention,the compounds of the present invention and the physiologicallyacceptable salts thereof, or the acid derivatives of either (hereinafterreferred to as the “active compound”) are typically admixed with, interalia, an acceptable carrier. The carrier must, of course, be acceptablein the sense of being compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier maybe a solid or a liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a tablet, which maycontain from 0.5% to 95% by weight of the active compound. One or moreof each of the active compounds may be incorporated in the formulationsof the invention, which may be prepared by any of the well-knowntechniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above).

In general, the formulations of the invention are prepared by uniformlyand intimately admixing the active compound with a liquid or finelydivided solid carrier, or both, and then, if necessary, shaping theresulting mixture. For example, a tablet may be prepared by compressingor molding a powder or granules containing the active compound,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the compound in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets may be made by molding, in a suitable machine,the powdered compound moistened with an inert liquid binder.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theactive compound, which preparations are preferably isotonic with theblood of the intended recipient. These preparations may be administeredby means of subcutaneous, intravenous, intramuscular, or intradermalinjection. Such preparations may conveniently be prepared by admixingthe compound with water or a glycine buffer and rendering the resultingsolution sterile and isotonic with the blood.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3(6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor bis\ris buffer (pH 6) or ethanol/water and contain from 0.01 to 0.2Mactive ingredient.

E. Methods of Use

In addition to the compounds of the formulas described herein, thepresent invention also provides useful therapeutic methods. For example,the present invention provides a method of inducing cytotoxicity againsttumor cells, antitumor-promoting activities, anti-inflammatory activity.More specifically, the present invention provides a method of inducingandrogen receptor antagonist activity. The androgen receptor antagonistactivity is a useful means of inhibiting androgen related tumor orcancer cell growth.

Cancer cells which may be inhibited include cells from skin cancer,small cell lung cancer, testicular cancer, lymphoma, leukemia,esophageal cancer, stomach cancer, colon cancer, breast cancer,endometrial cancer, ovarian cancer, central nervous system cancer, livercancer and prostate cancer.

The present invention also provides a method of treating cancer in asubject afflicted with cancer. These subjects also include subjectsafflicted with antiandrogen withdrawal syndrome. The method includesadministering to the subject in an effective cancer-treating amount acompound of the formulas of the present invention. The method is usefulfor the treatment of a variety of cancer cells which include but are notlimited to skin cancer, small cell lung cancer, testicular cancer,lymphoma, leukemia, esophageal cancer, stomach cancer, colon cancer,breast cancer, ovarian cancer, central nervous system cancer, livercancer and prostate cancer.

Compounds with anti-androgen activity also have the potential to betherapeutically useful for treatment of androgen-potentiated hairdisorders such as baldness and hirsutism. Anti-androgenic compounds mayalso be therapeutically useful as a form of male contraception where itis generally known and understood by those skilled in the art thatandrogens are required to maintain spermatogenesis. Additionally,compounds with anti-androgenic activity may be useful for the treatmentof behavioral disorders which include, but are not limited to,aggressiveness, violent behavior and sexual aggression. Anti-androgeniccompounds may also be therapeutically useful for the treatment of acnedue to the altered levels of hormones, including androgens, associatedwith acne disorders.

Subjects which may be treated using the methods of the present inventionare typically human subjects although the methods of the presentinvention may be useful for veterinary purposes with other subjects,particularly mammalian subjects including, but not limited to, horses,cows, dogs, rabbits, fowl, sheep, and the like. As noted above, thepresent invention provides pharmaceutical formulations comprising thecompounds of formulae described herein, or pharmaceutically acceptablesalts thereof, in pharmaceutically acceptable carriers for any suitableroute of administration, including but not limited to oral, rectal,topical, buccal, parenteral, intramuscular, intradermal, intravenous,and transdermal administration.

The therapeutically effective dosage of any specific compound will varysomewhat from compound to compound, patient to patient, and will dependupon the condition of the patient and the route of delivery. As ageneral proposition, a dosage from about 0.1 to about 50 mg/kg will havetherapeutic efficacy, with still higher dosages potentially beingemployed for oral and/or aerosol administration. Toxicity concerns atthe higher level may restrict intravenous dosages to a lower level suchas up to about 10 mg/kg, all weights being calculated based upon theweight of the active base, including the cases where a salt is employed.Typically a dosage from about 0.5 mg/kg to about 5 mg/kg will beemployed for intravenous or intramuscular administration. A dosage fromabout 10 mg/kg to about 50 mg/kg may be employed for oraladministration.

The present invention is explained in greater detail in the followingnon-limiting examples.

EXAMPLE 1

A. Materials and Methods

Compounds 1-3 were obtained by column chromatography (silica gel,CHCl₃-MeOH) of commercially available curcumin (Aldrich), whichcontained 2 and 3 as minor components. Compounds 39-44 were purchasedfrom Aldrich, Inc (Milwaukee, Wis.).

Dimethylcurcumin (4). Curcumin (1) in Et₂O and MeOH was treated withexcess of diazomethane in ether for 24 h. The solvents were removed invacuo and the residue was purified by silica gel column chromatographyand PLC to yield yellow needles of 4 (yield 19.8%); mp 129-130° C.(MeOH) (Roughley et al., J. Chem. Soc. Perkin I, 2379-2388(1973))(128-130° C.); ¹H NMR (300 MHz, CDCl₃): δ 3.93 (12H, s, OCH₃×4),5.82 (1H, s, 1-H), 6.48 (2H, d, 16 Hz), 6.88 (2H, d, J=8 Hz), 7.08 (2H,bs), 7.15 (2H, bd), 7.61 (2H, J=16 Hz); ¹³CNMR (300 MHz, CDCl₃): δ 55.9,56.0, 101.3, 109.8, 111.1, 122.0, 122.6, 128.1, 140.4, 149.2, 151.0,183.2.

Preparation of pyrazol derivative (8). To a solution of 1-4 in butanoland ethanol were added histidine hydrazide (1 equiv.), acetic acid andp-TsOH. The solution was refluxed for 24 h, and then the solvent wasremoved in vacuo. The residue was purified by silica gel columnchromatography and PLC.

Compound 8. Yellow powder (yield 17.5%), mp 166-168° C. (MeOH); ¹H NMR(300 MHz, CDCl 3): δ 3.92 (6H, s, OCH₃×2), 3.94 (6H, s, OCH₃×2), 6.62(1H, s, 1-H), 6.86 (2H, d, J=8 Hz), 6.93 (2H, d, J=16 Hz), 7.04 (2H, dd,J=8, 2 Hz), 7.06 (2H, bs), 7.05 (2H, d, J=16 Hz); ¹³CNMR (300 MHz,CDCl₃): δ 55.8, 55.9, 99.6, 108.6, 111.2, 115.8, 120.1, 129.7, 130.6,149.1, 149.3; Anal. calcd. for C₂₃H₂₄N₂O₄.1.¼H₂O: Theory: C, 66.57; H,6.44; N, 6.75. Found C, 66.44; H, 6.19; N, 6.27.

Monomethylcurcumin (9). Curcumin (1) in MeOH was treated with excessdiazomethane in Et₂O for 24 h. After removal of solvents, the residuewas purified by silica gel column chromatography and PLC to yield ayellow amorphous solid (yield 20%); mp 89-91° C., [α]_(D) −3.6 (c=1.14,CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 3.93 (9H, s, OCH₃×3), 5.81 (1H, s,1-H), 5.94 (1H, bs, OH), 6.49 (2 H, bd, J=15 Hz), 6.93 (1H, d, J=8 Hz),6.97 (1H, d, J=8 Hz), 7.10 (4H, m), 7.60 (2 H, bd, J=15 Hz); EIMS m/z382 (M*), HRFABMS 382.1396 (M+H⁺) (calcd for C₂₂H₂₂O₆: 382.1416).

Hydrogenation of 1, 4 and 10 (11-18). A solution of starting material inEtOAc was shaken with 10% Pd—C under H₂ (45 psi) overnight using aParr's apparatus. The solution was filtered and concentrated in vacuo togive a residue, which was purified by silica gel column chromatographyand PLC.

Tetrahydrocurcumin (11). White powder, mp 92-93° C. (Roughley et al., J.Chem. Soc. Perkin I, 2379-2388 (1973), 95-96° C.), ¹H NMR (300 MHz,CDCl₃): δ 2.53-2.58 (3H, m), 2.78-2.88 (5H, m), 3.87 (6H, s, OCH₃×2),5.43 (1H, s, 1-H), 5.50 (2H, s, ArOH), 6.65 (2H, d, J=8 Hz), 6.69 (2H,s), 6.83 (2H, d, J=8 Hz); ¹³CNMR (300 MHz, CDCl₃): δ 31.3, 40.4, 55.8,99.8, 111.0, 114.3, 120.8, 132.6, 144.0, 146.4 and 193.2.

Hexahydrocurcumin (12). White powder, mp 87-88° C. (Roughley, P. J. etal., J. Chem. Soc. Perkin I, 2379-2388 (1973), 78-80° C.), ¹H NMR (300MHz, CDCl₃): δ 1.60-1.81 (2H, m), 2.53-2.97 (8H, m), 3.85 (6H, s,OCH₃×2), 4.06 (1H, m, 2-H), 6.70 (4H, m), 6.80 (2H, d, J=8 Hz); ¹³CNMR(300 MHz, CDCl ₃): δ 29.7, 31.7, 38.8, 45.8, 49.8, 56.3, 67.4, 111.5,111.6, 114.8, 114.9, 121.2, 121.4, 133.0, 134.2, 144.2, 144.5, 146.9,147.9 and 211.9.

Octahydrocurcumin (13). Colorless oil, ¹H NMR (300 MHz, CDCl₃): δ 1.61(2H, m), 1.75 (4H, m), 2.53-2.70 (4H, m), 3.80 (6H, s, OCH₃×2), 3.91(2H, brs), 6.13 (2H, s, ArOH), 6.65 (2H, d, J=8 Hz), 6.69 (2H, bs) 6.82(2H, bd, J=8 Hz), ¹³CNMR (300 MHz, acetone-d₆): δ 31.1, 39.8, 42.6,35.6, 72.0, 111.0, 114.3, 120.6, 133.6, 143.6 and 146.4.

Compound 14. White powder (yield 26.0%), mp 60-61° C., ¹H NMR (300 MHz,CDCl₃): δ 2.56 (3H, m), 2.86 (5H, m), 3.85 (12H, s, OCH₃×4), 5.44 (1H,s, 1-H), 6.71 (4H, m), 6.78 (2H, bd); Anal. calcd. for C₂₃H₂₈O₆.¼H₂O:Theory: C 68.21; H, 7.09. Found C, 68.25; H, 7.06.

Compound 15. White powder (yield 20.0%), mp 94-95° C., ¹H NMR (300 MHz,CDCl₃): δ 1.65-1.80 (2H, m), 2.53-2.84 (8H, m), 3.85 (12H, s, OCH₃×4),4.05(1H, bs, 2′-H), 6.68-7.23 (4H, m), 6.79 (2H, bd), Anal. calcd. forC₂₃H₃₀O₆.¼H₂O: Theory: C, 67.88; H, 7.55. Found C, 67.73; H, 7.49.

Compound 16. Colorless oil (yield 4.2%), mp 60-61° C., ¹H NMR (300 MHz,CDCl₃): δ 1.55-1.65 (4H, m), 1.73-1.82 (3H, m), 2.60-2.72 (3H, m), 3.86(6H, s, OCH₃×2), 3.87 (8H, bs, OCH₃×2, 2,2′-H), 6.72-6.78 (4H, m), 6.79(2H, bd), 7.27(2H, s, OH×2), EIMS m/z: 404 (M⁺), HRFAB-MS m/z 404.219070(M+H)⁺ (calcd for C₂₃H₃₂O₆: 404.2198891).

Compound 17. Colorless oil (yield 5.9%), ¹H NMR (300 MHz, CDCl₃): δ 1.10(3H, d), 1.80 (1H, m), 2.43-2.82 (8H, m), 3.86 (6H, s, OCH₃×2), 3.87(6H, s, OCH₃×2), 3.94 (1H, bs, 2′-H)* 0.70-6.78 (6H, m), EIMS m/z416(M⁺).

Compound 18. Colorless oil (yield 6.95%), ¹H NMR (300 MHz, CDCl₃): δ0.95 (3H, d, 1-CH₃), 1.52 (1H, m), 1.84 (2H, m), 2.67 (6H, m), 3.83(14H, bs, OCH₃×4, 2, 2′-H), 6.78 (6H, m); EIMS m/z: 418 (M⁺), HRFAB-MSm/z 418.236618 (M+H)⁺(calcd for C₂₄H₃₄O₆: 418.2355392).

Preparation of 19 and 20. A mixture of curcumin (1, 100 mg, 0.81 mmol)in acetone (20 mL) with methylchloroacetate (2 mL) and NaI (20 mg) wasrefluxed with anhydrous potassium carbonate (176 mg) for 24 h withstirring. After filtration and removal of solvent, the residue waspurified by silica gel column chromatography to yield the correspondingmethyl acetates 19 and 20.

Compound 19: Yellow powder (yield 20.0%), mp 60-61° C., mp 66-67° C.,[α]_(D) −2.4 (c=2.08, CHCl₃); ¹H NMR (300 MHz, acetone-d₆): δ 3.73 (3H,s, —COOCH ₃), 3.86 (6H, s, OCH ₃×2), 4.79 (2H, s, O—CH ₂—COO), 5.99 (1H,s, 1-H), 6.70 and 6.73 (both 1H, d, J=15.3 Hz), 6.88 (1H, d, J=8 Hz),6.94 (1H, d, J=8 Hz), 7.17 (2H, m), 7.33 (2H, m), 7.59 and 7.61 (both1H, d, J=15.3 Hz), ¹³CNMR (300 MHz, CDCl₃): d 51.8, 55.9, 55.9, 65.9,101.4, 111.2, 111.6, 114.3, 115.9, 121.8, 122.6, 123.0, 123.5, 127.6,128.7, 129.8, 140.3, 141.3, 148.4, 149.8, 150.0, 150.4, 169.4, 183.4,184.6; Anal. calcd. for C₂₄H₂₄O₈.¾H₂O: Theory: C, 63.50; H, 5.66. FoundC, 63.53; H, 5.65.

Compound 20: Yellow powder (yield 20.0%), mp 141-142° C. (MeOH), [α]_(D)−0.29 (c=5.86, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 3.80 (6H, s), 3.93(6H, s), 4.73 (4H, s, O—CH ₂—COO×2), 5.82 (1H, s, 1-H), 6.50 (2H, d,J=16 Hz), 6.79 (2H, d, J=8 Hz), 7.09 (4H, bs), 7.58 (2H, d, J=16 Hz),¹³C NMR (300 MHz, CDCl₃): d 52.3, 56.0, 66.0, 101.4, 110.7, 113.6,122.0, 122.7, 129.5, 140.1, 149.0, 149.7, 169.0, 183.1; Anal. calcd. forC₂₇H₂₈O₁₀.½H₂O: Theory: C, 62.18; H, 5.60. Found C, 62.31; H, 5.57.

Compound 21: Yellow amorphous solid (yield 3.0%), ¹H NMR (300 MHz, CDCl₃): δ 2.58 (2H, m), 2.95 (2H, m), 7.12 (2H, d, J=15 Hz), 7.40 (6H, m),7.60 (4H, m), 7.81(2H, d, J=15 Hz), 12.65(1H, bs); Anal. calcd. forC₂₂H₂₀O₄: Theory: C, 75.84, H, 5.79. Found C, 75.56, H, 5.74.

Compound 22: Yellow amorphous solid (yield 25.0%), Anal. calcd. forC₂₆H₂₈O₈: Theory: C, 66.66, H, 6.02. Found C, 66.38, H, 6.16.

Compound 23: Yellow powder (yield 45.0%), mp 144-146° C. (MeOH)(Pedersen et al., Liebigs Ann. Chem. 1557-1569 (1985), 71-73° C.(CH₂Cl₂)) Anal. calcd. for C₂₄H₂₆O₈.2.½H ₂₀: Theory: C, 59.87; H, 5.23.Found C, 59.94; H, 5.11.

The structures of 1-4, 10-13, 22, and 23 were confirmed by comparison oftheir physical spectral data with those reported in the literature.Pedersen et al., Liebigs Ann. Chem. 1557-1569 (1985), Roughley et al.,J. Chem. Soc. Perkin I, 2379-2388 (1973).

B. Suppression of DHT-Mediated Transcription Activity.

Cell Culture and Transfections. Human prostate cancer DU145 and PC-3cells were maintained in Dulbecco's minimum essential medium (DMEM)containing penicillin (25 units/mL), streptomycin (25 μg/mL), and 10%fetal calf serum (FCS). For AR transactivation assay, PC-3 cells weretransfected with an AR expression plasmid and reporter gene. Because ofa low content of endogenous AR coactivators, DU-145 cells weretransfected with expression plasmids for AR and ARA70, and reportergene. The conditions were followed as previously described in Miyamotoet al., Proc. Natl. Acad. Sci. USA, 95, 7379-7384 (1998), with minormodifications.

Transfections were performed using the SuperFect kit according tomanufacturer's procedures (Qiagen, Chatsworth, Calif.). Briefly, 1×10⁵cells were plated on 35-mm dishes 24 h before transfection, and then areporter plasmid, MMTV-Luciferase, which contains MMTV-LTR promoter andAR-binding element, was co-transfected with an AR expression plasmid(wild type or mutant), or pSG5ARA70. PRL-TK was used as an internalcontrol for transfection efficiency. The total amount of DNA wasadjusted to 3.0 g with pSG5 in all transcriptional activation assays.After a 2 h transfection, the medium was changed to DMEM-10% charcoalstripped serum medium, and 14-16 h later, the cells were treated withDHT, antiandrogen, or test compounds. After another 14-16 h, the cellswere harvested and tested for luciferase activity in luciferase assays(Promega, Dual Luciferase Assay System, Madison, Wis.). Data wereexpressed in relative luciferase activity as compared to an internalluciferase positive control.

C. Results and Discussion

The aim of this work was to investigate novel curcumin analogues forantiandrogen receptor antagonist activity. The synthesis and evaluationof novel curcumin analogues as antiandrogen receptor antagonists andantitumor agents are reported herein.

Forty-seven curcumin derivatives (1-47) were tested for antagonisticactivity against the AR using two different human prostate cancer cells,PC-3 and DU-145 (FIGS. 3A-C). The parental compound, curcumin (1), wasinactive in all cases. However, dimethylated curcumin (4) showedsignificant antagonistic activity (reducing 70% of DHT-induced ARactivity) when assayed in PC-3 cells transfected with wild-type AR andwas more potent than HF (which reduced 16% of DHT-induced AR activity,FIG. 3A). Compound 4 also showed the highest antagonist activity whenassayed in DU-145 cells transfected with a mutant LNCaP AR and ARA70(showing a 45% reduction in DHT-induced AR activity, FIG. 3B),indicating that compound 4 is an effective antagonist for both normaland mutant AR.

To determine the structural requirements for AR antagonist activity inthis series of compounds, a structure-activity relationship (SAR) studywas conducted in a PC-3 cell assay system. Compared with 4,monomethylated curcumin (9) lacks one O-methyl groups at the p-positionon one benzene ring, and was significantly less active than 4 (FIG. 3B).Thus, the bis(3,4-dimethoxyphenyl) groups of 4 are important to theactivity. Introducing a methyl group at C-4 of 4 (10) resulted indecreased activity (FIG. 3B). Compounds 14 and 15, which were obtainedby hydrogenation of 4, were as potent as HF with an 18% reduction inDHT-induced AR activity, but were considerably less active compared to 4(FIG. 3A). Converting the β-diketone moiety of 4 to the correspondingpyrazol derivative 8 greatly reduced the activity. Furthermore,1,3-bis(3,4-dimethoxyphenyl)-1,3-propandione (39), which contains thebis-aryl groups found in 4 but lacks the conjugated double bonds, wasless active than 4 (FIGS. 3A and 3B), indicating that the conjugateddouble bonds also contribute to the activity of 4. These observationssuggested that the bis(3,4-dimethoxyphenyl) groups and the conjugatedβ-diketone moiety are crucial for the activity.

Data in FIG. 3C show a somewhat different cell assay system whereantiandrogen activity was assayed in DU-145 cells transfected withwild-type AR and ARA70. Compounds 4, 20, 22, 23, and 39 showedcomparable or more potent antiandrogen activity than HF in this assaysystem. Compounds 20 and 22 were almost equipotent (54% and 53.8%reduction, respectively) and were slightly more active than 4 (49.9%).Because curcumin (1) itself was not active, introducing eithermethoxycarbonylmethyl groups at the phenolic hydroxyls (20) or anethoxycarbonylethyl group at C-4 (22) greatly contributed to the anti-ARactivity in DU-145 cells in the presence of wild-type AR and ARA70.

In this study, we also examined the antiandrogen activity offluorodiarylheptanoids 24-29 and cyclic diarylheptanoids 30-38.Compounds 24-29 have fluorine or trifluoromethyl substituents on bothbenzene rings, but showed weak activity or were inactive. Among thecyclic diarylheptanoids 30-38, compound 30 was the most active and wasalmost as active as HF (FIGS. 3A and 3C). The remaining cyclicdiarylheptanoids showed weak antagonistic activity.

In conclusion, we have prepared a number of curcumin analogues andevaluated their potential antiandrogen activity in three different assayconditions using human prostate cancer cell lines. Compounds 4 showedpromising antiandrogen activities in all assays. Compounds 4, 20, 22, 23and 39 have been identified as a new class of antiandrogen agents. TheSAR study revealed that bis(3,4-dimethoxyphenyl) moieties, a conjugatedβ-diketone, and an ethoxycarbonylethyl group at the C-4 position playimportant roles in the antagonistic activity.

EXAMPLE 2 Synthesis and Characterization of Additional Curcumin Analogs

Additional curcumin analogs are shown in Table 1 below.

TABLE 1 Curcumin analogues. Compound Structure LL-7

LL-10

LL-17

LL-18

LL-27-B

LL-32-B

LL-35

LL-36

LL-41

LL-46

LL-55

LL-61

LL-62

LL-65

LL-66

LL-80

HOJ-7

HOJ-9

HOJ-10

The foregoing compounds are synthesized as follows:

LL-7: The synthetic method was modified from the strategy developed byPedersen et al. Acetylacetone (0.2 ml, 2 mmol) and boric anhydride (100mg, 1.4 mmol) were dissolved in 15 ml of ethyl acetate. The solution wasstirred at 70° C. for 0.5 hour. 5-hydroxymethylfuran (506 mg, 4 mmol)and tributyl borate (1.08 ml, 4 mmol) were added. The mixture wasstirred for 30 min. Then butylamine (0.3 ml, 3 mmol) dissolved in 4 mlof EtOAc was added dropwise during 15 min. The stirring continued for 5hours at 85° C. The mixture was then hydrolyzed by adding 8 ml of 1N HCland stirring for 0.5 hour at 60° C. The organic layer was separated. Theaqueous layer was extracted with ethyl acetate. The combined organiclayers were washed until neutral, dried with anhydrous sodium sulphate.The solven was removed in vacu. The crude products were purified byCombiFlash® column chromatography eluting with hexane-EtOAc. 68 mg redpowder was obtained in 12% yield. ¹H NMR (300 MHz, CDCl₃): δ 4.67 (4H,s), 5.74 (H, s), 6.40 (2H, d, J=3.3 Hz), 6.53 (2H, d, J=15.3 Hz), 6.58(2H, d, J=3.3 Hz), 7.43 (2H, d, J=15.6 Hz).

LL-10: vanillin (1.52 g, 10 mmol) and tributyl borate (2.7 ml, 10 mmol)were dissolved in 8 ml ethyl acetate. To the stirring solution, acetone(0.37 ml, 5 mmol) was added. The solution was stirring for 10 min.Butylamine (1 ml, 10 mmol) in 5 ml EA was added dropwise. The solutionwas stirring for hours at 50° C. 7 ml of 1N HCl was added. The mixturewas stirring for 0.5 hour at 50° C. ¹H NMR (300 MHz, CDCl₃): δ 3.94 (6H,s), 6.90 (2H, d, J=8.4 Hz), 6.95 (2H, d, J=15.6 Hz), 7.11 (2H, d, J=1.8Hz), 7.20 (2H, dd, J=1.8 Hz, J=8.4 Hz), 7.68 (2H,d, J=15.6 Hz).

LL-1: Acetylacetone (3.1 ml, 30 mmol) and boric anhydride (1.5 g, 21mmol) were dissolved in 20 ml of ethyl acetate. The solution was stirredat 70° C. for 1 hour. To the solution vanillin (1.52 g, 10 mmol) andtributyl borate (2.7 ml, 10 mmol) were added. The mixture was stirredfor 30 min. At 85° C., butylamine (1 ml, 10 mmol) dissolved in 7 ml ofEtOAc was added dropwise during 15 min. The stirring continued for 1hour at 100° C. The mixture was then hydrolyzed by adding 20 ml of 1NHCl at 50° C. and stirring for 0.5 hour at 50° C. The organic layer wasseparated. The aqueous layer was extracted with ethyl acetate. Thecombined organic layers were washed until neutral, dried with anhydroussodium sulphate. The solven was removed in vacu. The crude products werepurified by flash column chromatography eluting with hexane-EtOAc.Yellow powder was obtained in 49% yield. ¹H NMR (300 MHz, CDCl₃): δ 2.16(3H, s), 3.94 (3H, s), 5.63 (H, s), 6.33 (H, d, J=15.9 Hz), 6.92 (H, d,J=8.1 Hz), 7.01 (H, d, J=1.8 Hz), 7.10 (H, dd, J=8.1 Hz, J=1.8 Hz), 7.53(H, d, J=15.9 Hz).

LL-17: At 80° C., LL-1 (468, 2 mmol) and boric anhydride (100 mg, 1.4mmol) were dissolved in 10 ml of EtOAc. The solution was stirring for 1hour. To the mixture, 10 ml of EtOAc solution containing3,4-dimethoxybenzaldehyde (365 mg, 2.2 mmol) and tributyl borate (0.54ml, 2 mmol) was added. After stirring for 0.5 hour, piperidine (0.08 ml)was added to the mixture. After stirring for 2 hours, 4 ml of 0.4 N HClwas added at 50° C. The mixture was vigorously stirred at 50° C. for 0.5hour. The combined organic layers were washed until neutral, dried withanhydrous sodium sulphate. The solven was removed in vacu. The crudeproducts were purified to offer 67.2 mg LL-17 in 8.8% yield. ESI-MS m/z381.1 (M−1)⁺¹H NMR (300 MHz, CDCl₃): δ 3.94 (9H, s), 5.81 (H, s), 6.48(2H, d, J=12.6 Hz), 6.87-7.14 (6H, m), 7.60 (2H, d, J=12.6 Hz).

LL-18: the same procedure as the preparation of LL-17.50% yield (startedwith LL-1 and 4-hydroxybenzaldehyde). Orange powder. ESI-MS m/z 337.1(M−1)⁺; ¹H NMR (300 MHz, CD₃COCD₃): δ 3.93 (3H, s), 5.98 (H, s), 6.70(2H, d, J=15.9 Hz), 6.87-6.92 (3H, m), 7.19 (H, dd, J=8.4 Hz, J=1.8 Hz),7.35 (H, d, J=1.8 Hz), 7.56-7.64 (4H, m).

LL-27-B: Acetylacetone (3.1 ml, 30 mmol) and boric anhydride (1.5 g, 21mmol) were dissolved in 20 ml of ethyl acetate. The solution was stirredat 70° C. for 0.5 hour. 3-hydroxy-4-methoxybenzaldehyde (1.52 g, 10mmol) and tributyl borate (2.7 ml, 10 mmol) were added. The mixture wasstirred for 30 min at 70° C. Then butylamine (1 ml, 10 mmol) dissolvedin 7 ml of EtOAc was added dropwise during 15 min at 85° C. The stirringcontinued for 1 hour at 100° C. The mixture was then hydrolyzed byadding 20 ml of 1N HCl and stirring for 0.5 hour at 50° C. The organiclayer was separated. The aqueous layer was extracted with ethyl acetate.The combined organic layers were washed until neutral, dried withanhydrous sodium sulphate. The solvent was removed in vacu. The crudeproducts were purified. 1.04 g LL-27-A and 248 mg LL-27-B were obtained.ESI-MS m/z 367.1 (M−1)⁺; ¹H NMR (300 MHz, CD₃COCD₃): δ 4.01 (6H, s),6.01 (H, s), 6.66 (2H, d, J=15.6 Hz), 7.03 (2H, d, J=8.1 Hz), 7.20 (2H,dd, J=2.1 Hz, J=8.1 Hz), 7.26 (2H,d, J=2.1 Hz), 7.65 (2H, d, J=15.3 Hz).

LL-32-B: Acetylacetone (3.1 ml, 30 mmol) and boric anhydride (1.5 g, 21mmol) were dissolved in 20 ml of ethyl acetate. The solution was stirredat 70° C. for 0.5 hour. 2,3,4-trimethoxybenzaldehyde (1.96 g, 10 mmol)and tributyl borate (2.7 ml, 10 mmol) were added. The mixture wasstirred for 30 min at 70° C. Then butylamine (1 ml, 10 mmol) dissolvedin 7 ml of EtOAc was added dropwise during 15 min at 85° C. The stirringcontinued for 1 hour at 100° C. The mixture was then hydrolyzed byadding 20 ml of 1N HCl and stirring for 0.5 hour at 50° C. The organiclayer was separated. The aqueous layer was extracted with ethyl acetate.The combined organic layers were washed until neutral, dried withanhydrous sodium sulphate. The solvent was removed in vacu. The crudeproducts were purified. 1.25 g LL-32-A and 150 mg LL-32-B were obtained.¹H NMR (300 MHz, CDCl₃): δ 3.89 (6H, s), 3.90 (6H, s), 3.94 (6H, s),5.83 (H, s), 6.64 (2H, d, J=15.9 Hz), 6.71 (2H, d, J=9 Hz), 7.31 (2H, d,J=8.7 Hz), 7.85 (2H, d, J=16.2 Hz).

LL-35: The same procedure as the preparation of LL-17.55% yield (startedwith LL-32-A and 3,4-dimethoxybenzaldehyde). ESI-MS m/z 427.2 (M+H)⁺,449.3 (M+Na)⁺; ¹H NMR (300 MHz, CDCl₃): δ 3.89 (3H, s), 3.91 (3H, s),3.93 (3H, s), 3.94 (6H, s), 5.83 (H, s), 6.51 (H, d, J=15.9 Hz), 6.63(H, d, J=15.9 Hz), 6.71 (H, d, J=8.7 Hz), 6.89 (H, d, J=8.4 Hz), 7.09(H, d, J=2.1 Hz), 7.15 (H, dd, J=1.8 Hz, J=8.7 Hz), 7.31 (H, d, J=8.7Hz), 7.61 (H, d, J=15.9 Hz), 7.85 (H, d, J=16.2 Hz).

LL-36: The same procedure as the preparation of LL-17. 48% yield(started with LL-33-A and 3,4-dimethoxybenzaldehyde). ESI-MS m/z 427.2(M+H)⁺, 449.3 (M+Na)⁺; ¹H NMR (300 MHz, CDCl₃): δ 3.89(3H, s), 3.90 (3H,s), 3.93 (3H, s), 3.94 (3H, s), 3.95 (3H, s), 5.84 (H, s), 6.50 (H, d,J=15.6 Hz), 6.51(H, s), 6.57 (H, d, J=16.2 Hz), 6.88 (H, d, J=8.1 Hz),7.06 (H, s), 7.08 (H, d, J=2.1 Hz), 7.14 (H, dd, J=2.1 Hz, J=8.1 Hz),7.60 (H, d, J=15.9 Hz), 7.96 (H, d, J=15.9 Hz).

LL-41: To a solution of acetone (0.36 ml, 5 mmol) and3,4-dimethoxybenzaldehyde (1.66 g, 10 ml) in 50 ml of a 0.25 M solutionof aqueous NaOH was added 1.5 ml of a 25% w/w aqueous solution ofcetyltrimethylammonium bromide. The mixture was allowed to stirvigorously at room temperature for 20 hours, diluted with brine andextracted with ethyl acetate. The ethyl acetate solution wasconcentrated and then underwent column chromatography. 1.34 g brightyellow powder was obtained. 75% yield. ESI-MS m/z 355.2 (M+H)⁺; ¹H NMR(300 MHz, CD₃COCD₃): δ 3.94 (6H, s), 3.96 (6H, s), 6.90 (2H, d, J=8.4Hz), 6.95 (2H, d, J=15.9 Hz), 7.15 (2H, d, J=1.8 Hz), 7.20 (2H, dd,J=1.8 Hz, J=8.4 Hz), 7.69 (2H, d, J=15.9 Hz).

LL-46: The same procedure as the preparation of LL-17. 38% yield(started with LL-40 and 3,4-dimethoxybenzaldehyde). ESI-MS m/z 483.4(M+H)⁺; ¹H NMR (300 MHz, CDCl₃): δ 1.25 (3H, t, J=7.2 Hz), 2.32-2.37(2H, m), 2.55 (2H, t, J=7.8 Hz), 2.95 (0.5H, t, J=7.8 Hz), 3.91-3.97(9H, m), 4.14 (2H, q, J=7.2 Hz), 6.70 (H, dd, J=4.2 Hz, J=15.6 Hz),6.84-7.19 (7H, m), 7.62-7.76 (2H, m);

LL-55: To a stirred solution of LDA (0.29 ml, 2M hex/THF, 0.58 mmol) in3 ml of THF at −78° C. was added a solution of 3,4-dimethoxycinnamone(100 mg, 0.48 mmol) in THF (3 ml). After 15 min,3,4-dimethoxycinnamaldehyde (85 mg, 0.44 mmol) in THF (3 ml) was added.The mixture was stirred for 20 min at −78° C. And then the mixture wasquenched with saturated NH₄Cl. The solution was allowed to warm toambient temperature and extracted with ethyl acetate. 22 mg LL-55 wasobtained by column chromatography. 13% yield. ¹H NMR (300 MHz,CD₃COCD₃): δ 2.93 (2H, d), 3.80 (6H, s), 3.85 (6H, s), 4.13 (H, d), 6.25(H, dd, J=6 Hz, J=15.9 Hz), 6.58 (H, d, J=15.9 Hz), 6.80 (H, d, J=16.2Hz), 6.88 (H, d, J=8.7 Hz), 6.90 (H, dd, J=0.9 Hz, J=8.7 Hz), 7.00 (H,d, J=8.4 Hz), 7.04 (H, d, J=0.9 Hz), 7.25 (H, dd, J=0.9 Hz, J=8.7 Hz),7.34 (H, d, J=0.9 Hz), 7.61 (1H, d, J=16.2 Hz).

LL-61: 4-thoxycarbonylethyl curcumin (153.1 mg, 0.33 mmol) anddihydropyran (0.73 mL, 7.32 mmol) were dissolved in 3 mL of drydichloromethane containing PPTS (8.3 mg, 0.033 mmol). The solution wasstirred at room temperature for 48 hours. The solution was then washedby water. The solvent was removed in vacu. The crude products waspurified by CombiFlash® chromatography eluting with hexane-EtOAc to giveLL-61 (134 mg), 59% yield, yellow powder, mp 60-61° C.; ESI MS m/z 635.2(M−1)⁺; ¹H NMR (300 MHz, CDCl₃): δ 1.25 (3H, t), 1.57-2.17 (12H, m),2.96 (0.57H, t), 3.62 (4H, t), 3.91 (6H, s), 4.13 (2H, q), 5.47 (2H, t),6.72 (2H, d, J=15.6 Hz), 6.90-7.18 (6H, m), 7.44 (2H, d, J=15.6 Hz);

LL-62: A DMF solution (2 mL) of1,7-bis-(3,4-dimethoxyphenyl)-4-methyl-1,6-heptadiene-3,5-dione (50.3mg, 0.12 mmol) was added to an oil-free suspension of NaH (10 mg of 60%,6 mg, 0.4 mmol) in DMF (2 mL) under nitrogen at 0° C. The solution wasstirred at 0° C. for 30 min and then at room temperature for 2 h. To thesodium salt solution Selectfluoro™ (86.7 mg, 0.25 mmol) in DMF (2 mL)was added. After stirring for 2 h, the solution was extracted with EtOAcand washed with 5% H₂SO₄ (10 mL) and subsequently saturated NaHCO₃solution (10 mL). Then the solvent was evaporated in vacuo. The crudeproducts were purified by flash column chromatography eluting withhexane-EtOAc to afford LL-62 (25 mg) in 49% yield. yellow powder. ¹H NMR(300 MHz, CDCl₃): δ 1.97 (3H, s), 3.90 (6H, s), 3.95 (6H, s), 6.83 (2H,d, J=7.8 Hz), 6.87 (2H, dd, J=3 Hz, J=15.6 Hz), 6.90(2H, d, J=1.8 Hz),6.95 (2H, dd, J=1.8 Hz, J=7.8 Hz), 7.75 (2H, d, J=15.6 Hz);

LL-64B: Recrylstalized curcumin (1.08 g, 2.94 mmol) and dihydropyran (2mL, 20 mmol) were dissolved in 30 mL of dry dichloromethane containingPPTS (74 mg, 0.29 mmol). The solution was stirred at room temperaturefor 24 hours. The solution was then washed by water. The solvent wasremoved in vacuo. The crude products was purified by CombiFlash®chromatography eluting with hexane-EtOAc to give LL-64B (1.12 g), 66.8%yield, yellow powder; ESI MS m/z 535.0 (M−1)⁺; ¹H NMR (300 MHz, CDCl₃):δ 1.57-2.17 (12H, m), 3.62 (4H, t), 3.91 (6H, s), 5.47 (2H, t), 5.83(1H, s), 6.50 (2H, d, J=15.9 Hz), 7.09-7.16 (6H, m), 7.60 (2H, d, J=15.9Hz); Anal. Calcd (theoretical) for C₃₁H₃₆O₈.¼H₂O: C, 68.81; H, 6.80.Found: C, 68.86; H, 6.83.

LL-65: A THF solution (3 mL) of LL-64B (55 mg, 0.10 mmol) was added toan oil-free suspension of NaH (7 mg of 60%, 4.2 mg, 0.17 mmol) in THF (2mL) under nitrogen at 0° C. The solution was stirred at 0° C. for 30 minand then at room temperature for 2 h. To the sodium salt solution ethylpropiolate (0.02 mL, mmol) was added. After stirring for 2 h, thesolution was extracted with EtOAc and washed with 5% H₂SO₄ (10 mL) andsubsequently saturated NaHCO₃ solution (10 mL). Then the solvent wasevaporated in vacuo. The crude products were purified by CombiFlash®chromatography eluting with hexane-EtOAc to afford LL-65 (39.4 mg) 62%yield, orange powder; mp 72-73° C.; ESI MS m/z 634.7 M⁺; ¹H NMR (300MHz, CDCl₃): δ 1.34 (3H, t), 1.5-2.2 (12H, m), 3.62 (4H, t), 3.92 (6H,s), 4.28 (2H, q), 5.49 (2H, t) 5.96 (H, d, J=15.6 Hz), 7.00 (2H, d,J=15.6 Hz), 7.08-7.16 (6H, m), 7.76 (2H, d, J=15.3 Hz), 7.83 (H, d,J=15.9 Hz); Anal. Calcd (theoretical) for C₃₆H₄₂O₁₀1: C, 68.12; H, 6.67.Found: C, 67.82; H, 6.73.

LL-66: The EtOH (2.5 mL) solution of LL-65 (14.8 mg, 0.023 mmol) andPPTS (5 mg, 0.02 mmol) was stirred at room temperature for 3 h. Thesolution was evaporated in vacuo. LL-66 (10 mg) was obtained byCombiFlash® chromatography eluting with hexane-EtOAc. 93% yield, orangepowder, mp 106-106.5° C.; ESI MS m/z 465.2 (M−1)⁺; ¹H NMR (300 MHz,CDCl₃): δ 1.34 (3H, t), 3.95 (6H, s), 4.29 (2H, quart), 5.96 (2H, d,J=15.6 Hz), 6.95 (2H, d, J=8.2 Hz), 6.96 (1H, d, J=15.6 Hz), 7.05 (2H,d, J=2.1 Hz), 7.17 (2H, dd, J=8.2 Hz, J=2.1 Hz), 7.75 (2H, d, J=15.3Hz), 7.90 (1H, d, J=15.9 Hz).

LL-80: The same synthetic procedure as the preparation of LL-66. 62%yield (started with1,7-bis(3,4-dimethoxyphenyl)-1,6-pentadiene-3,6-dione). Red powder. ¹HNMR (300 MHz, CDCl₃): δ 1.34 (3H, t), 3.95 (12H, s), 4.29 (2H, quart),5.98 (2H, d, J=15.6 Hz), 6.95 (2H, d, J=8.4 Hz), 7.00 (1H, d, J=15.6Hz), 7.08 (2H, d, J=1.8 Hz), 7.22 (2H, dd, J=8.4 Hz, J=1.8 Hz), 7.77(2H, d, J=15.3 Hz), 7.91 (1H, d, J=15.6 Hz).

HOJ-7: 3% yield (started with 5 mmol of 2,4-difluorobenzaldehyde),yellow powder, mp 155.5-156° C. (n-hexane-EtOAc). ¹H NMR (CDCl₃) δ5.86(1H, s), 6.68 (2H, d, J=16.0 Hz), 6.84-6.96 (4H, m), 7.56 (2H, m), 7.71(2H, d, J=16.0 Hz). ESI-MS m/z 347 [M−1]⁺. Anal. calcd for C₁₉H₁₂F₄O₂:C, 65.52; H, 3.47. Found: C, 65.66; H, 3.57.

HOJ-9: 10% yield (started with 5 mmol of2-fluoro-4-methoxybenzaldehyde), yellow powder, mp 163-165° C.(n-hexane-EtOAc). ¹H NMR (CDCl₃) δ3.84 (6H, s), 5.82 (1H, s), 6.62 (2H,d, J=16.0 Hz), 6.70 (4H, m), 7.48 (2H, t, J=8.7 Hz), 7.71 (2H, d, J=16.0Hz). ESI-MS m/z 371 [M−1]⁺. Anal. calcd for C₂₁H₁₈F₂O₄: C, 67.74; H,4.87. Found: C, 67.58; H, 4.92.

HOJ-10: 9% yield (started with 5 mmol of2-fluoro-6-methoxybenzaldehyde), yellow needles, mp 144-145° C.(n-hexane-EtOAc). ¹H NMR (CDCl₃) δ3.82 (6H, s), 5.90 (1H, s), 6.72 (2H,d, J=16.0 Hz), 6.88 (2H, m), 7.56 (2H, m), 7.00-7.07 (4H, m), 7.74 (2H,d, J=16.0 Hz). ESI-MS m/z 371 [M−1]⁺. Anal. calcd for C₂₁H₁₈F₂O₄: C,67.74; H, 4.87. Found: C, 67.60; H, 4.95.

Biological activity. The compounds shown above are screened as followsand found to have anticancer and anti-androgen receptor activity asshown in Table 2 below.

Cell culture and transfection—Human prostate cancer LNCaP and PC-3 cellswere maintained in RPMI medium and Dulbecco's minimum essential medium(DMEM), respectively. Both media were strengthened with penicillin (25units/mL), streptomycin (25 μg/mL), and 10% fetal calf serum. Androgenreceptor transactivation assay, an androgen-dependent reporter genetranscription test, was employed as the primary screening for potentialantiandrogen identification. This assay was first performed in LNCaPcells, which express a clinically relevant mutant AR. Once potentialcompounds were indicated by LNCaP AR transactivation assay, they werere-examined for their potential activity against wild type AR. Wild typeAR transactivation assay was performed in PC-3 host cells, which lack anendogenous, functional AR.

Our previously described conditions for cell transfection were followed(Ohtsu et al, J Medicinal Chem 45: 5037-5042 (2002); Ohtsu et al.,Bioorganic & Medicinal Chemistry 11:5083-5090 (2003)). In brief, cellswere plated in 24- or 48-well tissue culture dishes twenty-four (PC-3cells) or forty-eight (LNCaP cells) hours prior to transfection.Subsequently, LNCaP cells were transfected with a reporter gene,MMTV-luciferase, which contains MMTV-LTR promoter and androgen receptorbinding element, and PRL-SV40, which served as an internal control fortransfection efficiency. PC-3 cells were transfected with a wild type ARexpression plasmid, pSG5AR, in addition to the above-mentionedMMTV-luciferase reporter gene and PRL-SV40 internal control. SuperFect(Qiagen, Chatsworth, Calif.) was employed as the transfection reagentfollowing manufacturer's recommendations. At the end of a five-hourtransfection, the medium was changed to DMEM or RPMI supplemented with10% charcoal dextran-stripped, androgen-depleted serum. Twenty-fourhours later, the cells were treated with 1 nM of DHT and/or testcompounds at the designated concentration for another twenty-four hours.The cells were harvested for luciferase activity assay using DualLuciferase Assay System (Promega, Madison, Wis.). The derived data wereexpressed as relative luciferase activity normalized to the internalluciferase control. DHT incubation induced a marked expression ofreporter gene. Test compounds capable of significantly suppressing thisandrogen-induced reporter gene expression were identified as potentialantiandrogens.

LNCaP cell growth assay: As mentioned above, LNCaP cells contain asubstantial amount of mutant AR, and thus these cells' growth can besignificantly activated upon androgen incubation. LNCaP cell growthassay thus was used as an alternative to confirm potential antiandrogensidentified by the above-mentioned AR transactivation assay. An MTTanalysis, relying upon the conversion of a colorless substrate toreduced tetrazolium by the mitochondrial dehydrogenase, was used. Theexperimental conditions were detailed elsewhere (Ohtsu et al.,Bioorganic & Medicinal Chemistry 11:5083-5090 (2003)). Briefly, cellswere plated in 96-well tissue culture plates and then incubated for 5consecutive days in the presence of 1 nM of DHT and/or test compounds in10% charcoal dextran-stripped serum-containing RPMI. At the end ofincubation, the cells were given MTT (5 mg/ml in PBS) for three hours at37° C. The resultant precipitate was dissolved in a lysis buffer andthen quantitated at a wavelength of 595 nm using a microplate reader. Toensure the accuracy of data derived from the MTT analysis, cell countwas performed using duplicate samples. Test compounds that displayed anadverse effect on the androgen-induced prostate tumor cell growth wereidentified as potential antiandrogens.

TABLE 2 The anti-AR activity of curcumin analogues. Compound wrAR/PC3LNCaP LL-7 0 + LL-10 0 + LL-17 + 0 LL-18 + 0 LL-27B + 0 LL-32B + 0LL-35 + 0 LL-36 + 0 HOJ-7 + + HOJ-9 + 0 HOJ-10 + + LL-41 + + LL-46 + +LL-55 0 + LL-61A + + LL-62 0 + LL-65/LL-80 ++ ++ LL-66 0 +

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A compound according to formula II:

wherein: R₁₁ and R₁₂ are each independently selected from the group consisting of alkoxy, nitro, amino, and dialkylamino; R₁₃ and R₁₄ are each independently selected from the group consisting of hydroxy, alkoxy, —OR₁₈C(O)R₁₉ wherein R₁₈ is lower alkylene or alkenylene and R₁₉ is alkoxy, and tetrahydropyranyl[THP]; or R₁₁ and R₁₃ together are alkylenedioxy; or R₁₂ and R₁₄ together are alkylenedioxy; R₁₅ and R₁₆ are each independently selected from the group consisting of H, halogen, and nitro; R₁₇ is —R₂₀C(O)OR₂₁, wherein R₂₀ is alkylene or alkenylene and R₂₁ is H or alkyl; X₃ is N, or X₃ is C bonded to a H, alkoxy or nitro; and X₄ is N, or X₄ is C bonded to a H, alkoxy or nitro; or a pharmaceutically acceptable salt thereof, subject to the proviso that R₂₀ is not alkylene when R₁₁, R₁₂, R₁₃ and R₁₄ are methoxy, and X₃ and X₄ are C bonded to a methoxy.
 2. The compound according to claim 1, wherein R₁₁ and R₁₂ are each independently selected from the group consisting of alkoxy, nitro, amino, and dimethylamino.
 3. The compound according to claim 1, wherein R₁₁ and R₁₃ together are methylenedioxy or ethylenedioxy.
 4. The compound according to claim 1, wherein R₁₂ and R₁₄ together are methylenedioxy or ethylenedioxy.
 5. The compound according to claim 1, wherein R₁₅ and R₁₆ are each independently selected from the group consisting of H, F, and nitro.
 6. A pharmaceutical formulation comprising a compound according to claim 1 in a pharmaceutically acceptable carrier.
 7. The pharmaceutical formulation according to claim 6, wherein said carrier is an aqueous carrier.
 8. The compound according to claim 1 having the structure:


9. The compound according to claim 1 having the structure: 